Decellularization and recellularization of organs and tissues

Abstract

To provide methods and materials for decellularizing an organ or portion thereof and recellularizing such a decellularized organ or portion thereof to thereby generate an organ or portion thereof.SOLUTION: A method for decellularization and recellularization of organs and tissues and an administration method therefor include a decellularized extracellular matrix including an exterior surface. The extracellular matrix, including the vascular tree, substantially retains the morphology of the extracellular matrix prior to decellularization. The exterior surface is substantially intact.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to organs and tissues, and more particularly to methods and materials for decellularizing and recellularizing organs and tissues.

Background Art

[0002] Background Biologically-derived matrices have been developed for tissue engineering and regeneration. However, the matrices developed to date generally have a damaged matrix structure and / or do not exhibit a vascular bed that can effectively reconstruct an organ or tissue. The present disclosure describes methods for decellularizing and recellularizing organs and tissues.

Summary of the Invention

[0003] Summary The present disclosure provides methods and materials for decellularizing an organ or tissue, and methods and materials for recellularizing a decellularized organ or tissue.

[0004] In one aspect, a decellularized mammalian heart is provided. The decellularized mammalian heart includes a decellularized extracellular matrix of the heart having an outer surface. The extracellular matrix of the decellularized heart substantially retains the morphology of the extracellular matrix before decellularization, and the outer surface of the extracellular matrix is substantially intact.

[0005] Exemplary hearts include, but are not limited to, rodent hearts, porcine hearts, rabbit hearts, bovine hearts, ovine hearts, or canine hearts. Another exemplary heart is a human heart. The decellularized heart may be a cadaveric heart. In some embodiments, the decellularized heart is a part of the whole heart. For example, parts of the whole heart can include, but are not limited to, a heart patch, aortic valve, mitral valve, pulmonary valve, tricuspid valve, right atrium, left atrium, right ventricle, left ventricle, septum, coronary vasculature, pulmonary artery, or pulmonary vein.

[0006] In another aspect, a solid organ is provided. The solid organs described herein include the decellularized heart and the collection of regenerative cells attached thereto. In some embodiments, the regenerative cells are pluripotent cells. In some embodiments, the regenerative cells are embryonic stem cells, umbilical cord cells, adult-derived stem or progenitor cells, bone marrow-derived cells, blood-derived cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, multipotent adult progenitor cells (MAPCs), cardiac stem cells (CSCs), or multipotent adult heart-derived stem cells. In some embodiments, the regenerative cells are cardiac fibroblasts, cardiac microvascular system cells, or aortic endothelial cells. In some embodiments, the cells are tissue-derived cells, or skin-derived cells.

[0007] Generally, the number of regenerative cells attached to the decellularized heart is at least about 1,000. In some embodiments, the number of regenerative cells attached to the decellularized heart is from about 1,000 cells / mg of tissue (wet weight; i.e., weight before decellularization) to about 10,000,000 cells / mg of tissue (wet weight). In some embodiments, the regenerative cells are heterologous to the decellularized heart. In some embodiments, the solid organ is for transplantation into a patient and the regenerative cells are autologous to the patient.

[0008] In yet another aspect, a method of making a solid organ is provided. Such a method generally includes the step of obtaining a decellularized heart as described herein, and contacting the decellularized heart with a collection of regenerative cells under conditions in which the regenerative cells will engraft, proliferate, and / or differentiate within and on the decellularized heart. In one embodiment, the regenerative cells are injected or perfused into the decellularized heart.

[0009] In yet another aspect, a method of decellularizing a heart is provided. Such a method includes obtaining a heart, cannulating the heart in one or more cavities, tubes, and / or ducts to create a cannulated heart, and perfusing the cannulated heart with a first cell disruption medium via one or more of the cannulations. For example, perfusion can be performed in multiple directions from each of the cannulated cavities, tubes, and / or ducts. Typically, the cell disruption medium includes at least one surfactant such as SDS, PEG, or Triton X.

[0010] Also, such a method can include perfusing the cannulated heart with a second cell disruption medium via two or more of the cannulations. Generally, the first cell disruption medium is an anionic surfactant such as SDS, and the second cell disruption medium can be an ionic surfactant such as Triton X-100. In such a method, perfusion can take about 2 to 12 hours per gram (wet weight) of heart tissue.

[0011] In one aspect, a solid organ is provided. Such a solid organ includes a decellularized organ and a collection of regenerated cells attached thereto. Such a decellularized organ includes the decellularized extracellular matrix of the organ, the extracellular matrix includes an outer surface, and the extracellular matrix including the vascular tree substantially retains the morphology of the extracellular matrix before decellularization, and the outer surface is substantially intact.

[0012] Representative solid organs include the heart, kidney, liver, or lung. In one aspect, the solid organ is the liver or a part of the liver. In another aspect, the solid organ is the heart (e.g., a rodent heart, a pig heart, a rabbit heart, a bovine heart, a sheep heart, or a dog heart; e.g., a heart showing contractile activity). A representative heart is a human heart. The heart can be a part of the whole heart (e.g., the aortic valve, the mitral valve, the pulmonary valve, the tricuspid valve, the right atrium, the left atrium, the right ventricle, the left ventricle, a heart patch, the septum, the coronary vessels, the pulmonary artery, and the pulmonary vein). In another aspect, the solid organ is the kidney. The solid organs described herein typically include a plurality of histological structures including blood vessels.

[0013] In some aspects, the number of regenerative cells attached to the decellularized organ is at least about 1,000. In other aspects, the number of regenerative cells attached to the decellularized organ is from about 1,000 cells / mg tissue to about 10,000,000 cells / mg tissue. The regenerative cells may be pluripotent cells. Alternatively, the regenerative cells may be embryonic stem cells or a subset thereof, umbilical cord cells or a subset thereof, bone marrow cells or a subset thereof, peripheral blood cells or a subset thereof, adult-derived stem cells or progenitor cells or a subset thereof, tissue-derived stem cells or progenitor cells or a subset thereof, mesenchymal stem cells (MSCs) or a subset thereof, skeletal muscle-derived stem cells or progenitor cells or a subset thereof, multipotent adult progenitor cells (MAPCs) or a subset thereof, cardiac stem cells (CSCs) or a subset thereof, or multipotent adult heart-derived stem cells or a subset thereof. Examples of regenerative cells include cardiac fibroblasts, cardiac microvascular endothelial cells, aortic endothelial cells, or hepatocytes. In some aspects, the regenerative cells are autologous or allogeneic or xenogeneic to the decellularized organ.

[0014] In some aspects, the solid organ is to be transplanted into a patient and the regenerative cells are autologous to the patient. In other aspects, the solid organ is to be transplanted into a patient and the decellularized organ is allogeneic or xenogeneic to the patient.

[0015] In another aspect, a method of manufacturing an organ is provided. Such a method generally includes a step of obtaining a decellularized organ, wherein the decellularized organ includes a decellularized extracellular matrix of the organ, the extracellular matrix includes an outer surface, and the extracellular matrix including a vascular tree substantially retains the morphology of the extracellular matrix before decellularization, and the outer surface is substantially intact; and a step of contacting the decellularized organ with a collection of regenerative cells under conditions where the regenerative cells are seeded, proliferated, and / or differentiated in or on the decellularized organ. In one aspect, the regenerative cells are injected into the decellularized organ. Representative decellularized organs include the heart, kidney, liver, spleen, pancreas, or lung.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Also, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0017] The following lists the basic features and various aspects of the present invention. [1] A step of providing a decellularized liver, wherein the decellularized liver includes a decellularized extracellular matrix of the liver, the extracellular matrix includes an outer surface, the extracellular matrix substantially retains the morphology of the extracellular matrix before decellularization including a vascular tree, and the outer surface is substantially intact; and a step of contacting the decellularized liver with about 40,000 or more regenerative cells under conditions where the cells are seeded, proliferated, and / or differentiated in and on the decellularized liver A method of manufacturing a liver, comprising: [2] The method according to [1], wherein the decellularized liver is brought into contact with about 23 million or more regenerative cells. [3] The method according to [1], wherein the decellularized liver is brought into contact with about 30 million or more regenerative cells. [4] The method according to [1], wherein the decellularized liver is brought into contact with about 35 million or more regenerative cells. [5] The method according to [1], wherein the regenerative cells are hepatocytes. [6] The method according to [1], wherein the regenerative cells are injected into the decellularized liver via the portal vein. [7] The method according to [1], wherein the regenerative cells are injected into the decellularized liver. [8] A step of providing a decellularized liver or a lobe-containing portion thereof, wherein the decellularized liver or the lobe-containing portion thereof comprises a decellularized extracellular matrix of the liver or the lobe-containing portion thereof, the extracellular matrix comprises an outer surface, the extracellular matrix such as a vascular tree substantially retains the morphology of the extracellular matrix before decellularization, and the outer surface is substantially intact, said step, A step of bringing a lobe of the decellularized liver or a lobe-containing portion thereof into contact with a collection of regenerative cells under conditions such that the regenerative cells engraft, proliferate and / or differentiate within and on the decellularized liver lobe A method for producing a liver lobe, comprising: [9] The method according to [8], wherein the regenerative cells are primary hepatocytes.

[10] The method according to [8], wherein the regenerative cells are injected into the lobe via the portal vein.

[11] A step of providing an organ; A step of inserting a cannula into one or more cavities, tubes, and / or ducts of the organ to produce a cannula-inserted organ; A step of perfusing a first cell-destruction medium into the cannula-inserted organ via the one or more cannula insertions; A step of determining the amount of nucleic acid remaining in the decellularized organ compared to the corresponding cadaveric organ, and A method for decellularizing an organ, comprising:

[12] The method according to

[11] , wherein the perfusion is about 2 to 12 hours per gram of organ tissue.

[13] The method according to

[11] , wherein the perfusion step is continued until the nucleic acid in the decellularized organ is 5% or less.

[14] The method according to

[11] , wherein the cell disruption medium contains 1% SDS.

[15] The method according to

[11] , wherein the perfusion is performed in multiple directions from each of the cannula-inserted cavities, tubes, and / or conduits.

[16] A decellularized mammalian adrenal gland comprising a decellularized extracellular matrix of the adrenal gland, wherein the extracellular matrix includes an outer surface, the extracellular matrix such as a vascular tree substantially retains the morphology of the extracellular matrix before decellularization, and the outer surface is substantially intact. The decellularized mammalian adrenal gland according to the above. Details of one or more aspects of the present invention are shown in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will become apparent from the drawings, the detailed description, and the claims.

Brief Description of the Drawings

[0018]

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[0019] Like reference numerals in different drawings indicate like components.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description Solid organs generally have three main components: an extracellular matrix (ECM), cells embedded therein, and a vascular bed. Decellularization of the solid organs described herein removes most or all of the cellular components while substantially preserving the extracellular matrix (ECM) and the vascular bed. Therefore, the decellularized solid organs can be used as scaffolds for recellularization. Mammals from which solid organs can be obtained include, but are not limited to, rodents, pigs, rabbits, cows, sheep, dogs, and humans. Organs and tissues used in the methods described herein may be cadavers or fetuses, neonates, or adults.

[0021] Solid organs referred to in this specification include, but are not limited to, the heart, liver, lungs, skeletal muscle, brain, pancreas, spleen, kidneys, stomach, uterus, and bladder. As used herein, a solid organ refers to an organ having a "substantially closed" vascular system. By a "substantially closed" vascular system with respect to an organ is meant that, assuming the major blood vessels have been cannulated, ligated, or otherwise constrained, during perfusion with a liquid, most of the liquid is contained within the solid organ and does not leak outside the solid organ. Despite having a "substantially closed" vascular system, many of the solid organs listed above have distinct "inlet" and "outlet" tubes that are useful for introducing and moving liquid throughout the organ during perfusion.

[0022] In addition to the above solid organs, for example, an entire or a part of a joint (e.g., knee, shoulder, waist or spine), trachea, skin, mesentery or intestine, small intestine, large intestine, esophagus, ovary, penis, testis, spinal cord, or an organ or tissue having other types of blood vessels such as a single blood vessel or a branched blood vessel can be decellularized using the methods disclosed herein. Further, the methods disclosed herein can also be used to decellularize avascular (or relatively avascular) tissues such as cartilage or cornea.

[0023] The decellularized organs or tissues (e.g., heart or liver) described herein or any part thereof (e.g., aortic valve, mitral valve, pulmonary valve, tricuspid valve, pulmonary vein, pulmonary artery, coronary vasculature, septum, right atrium, left atrium, right ventricle, left ventricle or liver lobe) can be used for transplantation into a patient, regardless of the presence or absence of recellularization. Alternatively, the recellularized organs or tissues described herein can be used, for example, to examine cells and / or cell organization during the differentiation of an organ or tissue.

[0024] Decellularization of Organs or Tissues The present invention provides methods and materials for decellularizing mammalian organs or tissues. The first step for decellularizing an organ or tissue is, if possible, to insert a cannula into the organ or tissue. Cannulas can be inserted into the tubes, ducts, and / or cavities of the organ or tissue using methods and materials known in the art. The next step for decellularizing the organ or tissue is to perfuse the cannula-inserted organ or tissue with a cell disruption medium. The perfusion across the organ can be multi-directional (e.g., anterograde and retrograde).

[0025] Langendorff perfusion of the heart (also known as four chamber working mode perfusion) is a conventional technique in the art as a physiological perfusion. See, for example, Dehnert, The Isolated Perfused Warm-blooded Heart According to Langendorff, in Methods in Experimental Physiology and Pharmacology: Biological Measurement Techniques V. Biomesstechnik-Verlag March GmbH, West Germany, 1988. Briefly, for Langendorff perfusion, a cannula is inserted into the aorta and attached to a reservoir containing the cell disruption medium. The cell disruption medium can be delivered in a retrograde direction to the aorta and is delivered at a constant flow rate, for example, by injection or a roller pump or by a certain hydrostatic pressure. In either case, the aortic valve is forced closed, directing the perfusion fluid towards the coronary ostia (thereby perfusing the entire ventricles of the heart), and then this flows out into the right atrium via the coronary venous ostia. For working mode perfusion, a second cannula can be connected to the left atrium and the perfusion can be changed from retrograde to anterograde.

[0026] Methods of perfusing other organs or tissues are known in the art. As examples, the following references describe perfusion of the lung, liver, kidney, brain, and limb. Van Putte et al, 2002, Ann. Thorac. Surg., 74(3):893-8; den Butter et al., 1995, Transpl. Int., 8:466-71; Firth et al., 1989, Clin. Sd.(Lond.), 77(6):657-61; Mazzetti et al., 2004, Brain Res., 999(l):81-90; Wagner et al., 2003, J.Artif. Organs, 6(3):183-91.

[0027] One or more cell disruption media can be used to decellularize an organ or tissue. The cell disruption media generally includes at least one surfactant such as SDS, PEG, or Triton X. The cell disruption media can include water such that the media is not osmotically compatible with the cells. Alternatively, the cell disruption media can include a buffer (e.g., PBS) for osmotic compatibility with the cells. The cell disruption media can also include enzymes such as one or more collagenases, one or more dispases, one or more DNases, or proteases such as trypsin (but not limited to these). In some cases, the cell disruption media can include, simultaneously or alternatively, one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors and / or collagenase inhibitors).

[0028] In certain embodiments, two different cell disruption media can be perfused sequentially through the cannulated organ or tissue. For example, the first cell disruption media can include an anionic surfactant such as SDS, and the second cell disruption media can include an ionic surfactant such as Triton X-100. After perfusion with at least one cell disruption media, the cannulated organ or tissue can be perfused with, for example, a wash solution and / or a solution containing one or more enzymes as disclosed herein.

[0029] Alternating the direction of perfusion (e.g., anterograde and retrograde) can help to effectively decellularize an entire organ or tissue. The decellularization described herein essentially completely decellularizes the organ to the inside with little damage to the ECM. The organ or tissue can be decellularized at an appropriate temperature of 4 to 40 °C. Depending on the size and weight of the organ or tissue, a particular (one or more) surfactant, and the concentration of the (one or more) surfactant in the cell disruption medium, the organ or tissue is generally perfused with the cell disruption medium for about 2 to about 12 hours per gram of solid organ or tissue. The organ may be perfused for up to about 12 to about 72 hours per gram of tissue, including washing. Perfusion is generally adjusted to physiological conditions including pulsatile flow, pulsation rate, and pulsation pressure.

[0030] As shown herein, a decellularized organ or tissue is composed essentially of the extracellular matrix (ECM) components of all or most regions of the organ or tissue, including the ECM components of the vascular tree. The ECM components can include any or all of the following: fibronectin, fibrin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, substrates, reticular fibers, and thrombospondin. These can remain organized as distinct structures such as the basement membrane. The outcome of decellularization is defined by the absence of detectable muscle filaments, endothelial cells, smooth muscle cells, and nuclei in histological sections using standard histological staining procedures. It is preferred but not essential that residual cell debris has also been removed from the decellularized organ or tissue.

[0031] To effectively recellularize and create an organ or tissue, it is important that the morphology and structure of the ECM are maintained (i.e., remain substantially intact) during or after the decellularization process. As used herein, "morphology" refers to the overall shape of the organ or tissue, or the ECM, and "structure" as used herein refers to the outer surface, inner surface, and the ECM therebetween.

[0032] The form and structure of the ECM can be examined visually and / or histologically. For example, the basement membrane on the outer surface of a solid organ or within the vasculature of an organ or tissue should not be removed or significantly damaged by decellularization. Further, the fibrils of the ECM should be similar to or not significantly different from those of the non-decellularized organ or tissue. Unless otherwise specified, decellularization as used herein refers to perfusion decellularization, and unless otherwise specified, the decellularized organs or matrices referred to herein are obtained using the perfusion decellularization described herein. The perfusion decellularization described herein can be compared, for example, to the immersion decellularization described in U.S. Patent Nos. 6,753,181 and 6,376,244.

[0033] One or more compounds can be applied onto or within a decellularized organ or tissue to, for example, preserve the decellularized organ, prepare the decellularized organ or tissue for recellularization, and / or assist or stimulate cells during the recellularization process. Such compounds include, but are not limited to, one or more growth factors (e.g., VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, and HGF), immunomodulators (e.g., cytokines, glucocorticoids, IL2R antagonists, leukotriene antagonists), and / or factors that modify the coagulation cascade (e.g., aspirin, heparin-binding proteins, and heparin). Also, the decellularized organ or tissue can be further treated, for example, by irradiation (e.g., UV, gamma) to reduce or eliminate the presence of any type of microorganism remaining on or within the decellularized organ or tissue.

[0034] Recellularization of Organs or Tissues The present invention provides materials and methods for generating organs or tissues. Organs or tissues can be generated by contacting the decellularized organs or tissues described herein with a collection of regenerative cells. The regenerative cells used herein are any cells used to recellularize the decellularized organs or tissues. The regenerative cells may be totipotent cells, pluripotent cells, or multipotent cells, and may be uncommitted or committed. The regenerative cells may also be single-lineage cells. Furthermore, the regenerative cells may be undifferentiated cells, partially differentiated cells, or fully differentiated cells. Examples of regenerative cells used herein include embryonic stem cells (as defined by the National Institutes of Health (NIH); see, e.g., the glossary at stemcells.nih.gov on the World Wide Web). Also included as regenerative cells are progenitor cells, precursor cells, and "adult"-derived stem cells including umbilical cord cells and fetal stem cells.

[0035] Examples of regenerative cells that can be used to recellularize an organ or tissue include, but are not limited to, embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow-derived stem or progenitor cells, blood-derived stem or progenitor cells, adipose tissue-derived stem or progenitor cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, or multipotent adult progenitor cells (MAPCs). Further regenerative cells that can be used include tissue-specific stem cells including cardiac stem cells (CSCs), pluripotent adult heart-derived stem cells, cardiac fibroblasts, cardiac microvascular endothelial cells, or aortic endothelial cells. Bone marrow-derived stem cells such as bone marrow mononuclear cells (BM-MNCs), endothelial or vascular-derived stem or progenitor cells, and peripheral blood-derived stem cells such as endothelial progenitor cells (EPCs) can also be used as regenerative cells.

[0036] The number of regenerative cells introduced into or onto a decellularized organ to generate an organ or tissue depends on both the organ (e.g., organ type, organ size and weight) or tissue, as well as the type and developmental stage of the regenerative cells. Different types of cells may have different tendencies regarding the aggregate density they reach. Similarly, different organs or tissues may be recellularized at different densities. By way of illustration, a decellularized organ or tissue can be “seeded” with at least about 1,000 (e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000) regenerative cells; or about 1,000 cells / mg of tissue (wet weight, i.e., before decellularization) to about 10,000,000 cells / mg of tissue (wet weight) can be attached.

[0037] Regenerative cells can be introduced (“seeded”) at one or more locations into a decellularized organ or tissue by injection. Further, two or more types of cells (i.e., a cocktail of cells) can be introduced into a decellularized organ or tissue. For example, the cocktail of cells can be injected at multiple locations of the decellularized organ or tissue, or different cell types can be injected into different parts of the decellularized organ or tissue. Instead of or in addition to injection, the regenerative cells or the cocktail of cells can be introduced into the cannulated decellularized organ or tissue by perfusion. For example, a perfusion medium can be used to perfuse the regenerative cells into the decellularized organ, and the medium can be changed to an expansion and / or differentiation medium to induce growth and / or differentiation of the regenerative cells.

[0038] During recellularization, the organ or tissue is maintained under conditions in which at least a portion of the regenerative cells can proliferate and / or differentiate within or on the decellularized organ or tissue. These conditions include appropriate temperature and / or pressure, electrical and / or mechanical activity, force, an appropriate amount of O 2 and / or CO 2, suitable amounts of humidity, as well as sterilization or near-sterilization conditions, among others, but not limited thereto. During recellularization, the decellularized organ or tissue, and the regenerative cells attached thereto, are maintained in a suitable environment. For example, the regenerative cells may require nutrient supplements (e.g., nutrients and / or a carbon source such as glucose), exogenous hormones or growth factors, and / or a specific pH.

[0039] The regenerative cells may be autologous to the decellularized organ or tissue (e.g., seeding human regenerative cells onto a human decellularized organ or tissue), or the regenerative cells may be heterologous to the decellularized organ or tissue (e.g., seeding human regenerative cells onto a porcine decellularized organ or tissue). As used herein, "autologous" refers to cells obtained from the same species as the species from which the organ or tissue is derived (e.g., oneself (i.e., autologous), or related or unrelated individuals), and "heterologous" as used herein refers to cells obtained from a species different from the species from which the organ or tissue is derived.

[0040] In some cases, the organs or tissues generated by the methods described herein are those to be transplanted into a patient. In this case, the regenerative cells used to recellularize the decellularized organ or tissue can be obtained from the patient such that the regenerative cells are "autologous" to the patient. Patient-derived regenerative cells can be obtained, for example, from blood, bone marrow, tissue, or organs at different life stages (e.g., prenatally, neonatally or perinatally, during adolescence, or as an adult) using methods known in the art. Alternatively, the regenerative cells used to recellularize the decellularized organ or tissue may be syngeneic (i.e., from a monozygotic twin) to the patient, the regenerative cells may be, for example, human lymphocyte antigen (HLA)-matched cells from a blood relative of the patient or an HLA-compatible individual unrelated to the patient, and the regenerative cells may be, for example, allogeneic to the patient from a non-HLA-compatible donor.

[0041] Regardless of the origin of the regenerative cells (e.g., whether autologous or not), the decellularized solid organ can be autologous, allogeneic or heterologous to the patient.

[0042] In certain examples, the decellularized organ can be recellularized in vivo by cells (e.g., after the organ or tissue has been transplanted into an individual). In vivo recellularization can be performed, for example, with any of the regenerative cells described herein, as described above (e.g., injection and / or perfusion). Alternatively or additionally, in vivo seeding of endogenous cells into the decellularized organ or tissue can occur naturally or be mediated by factors delivered to the recellularized tissue.

[0043] The progression of the regenerative cells can be monitored during recellularization. For example, the number of cells on or in the organ or tissue can be evaluated by performing a biopsy at one or more time points during recellularization. Additionally, the degree of differentiation the regenerative cells have undergone can be monitored by determining whether various markers are present in the cells or cell aggregates. Markers associated with different cell types and different differentiation stages of those cell types are known in the art and can be readily detected using antibodies and standard immunoassays. See, for example, Current Protocols in Immunology, 2005, Coligan et al, Eds., John Wiley & Sons, Chapters 3 and 11. Nucleic acid assays, as well as morphological and / or histological evaluations, can be used to monitor recellularization. Functional analysis of the recellularized organ can also be evaluated. For example, in a recellularized heart, contraction and intraventricular pressure can be evaluated, in a recellularized liver, albumin production, urea production, and cytochrome p450 activity can be evaluated, in a recellularized kidney, blood or media filtration and urine production can be evaluated, in a recellularized pancreas, blood, glucose, and insulin can be evaluated, in recellularized muscle, force generation or response to stimulation can be evaluated, and in recellularized vessels, thrombosis can be evaluated.

[0044] Control System for Decellularizing and / or Recellularizing Organs or Tissues The present invention also provides a system (e.g., a bioreactor) for decellularizing and / or recellularizing an organ or tissue. Such a system generally includes at least one cannula insertion device for inserting a cannula into the organ or tissue, a perfusion device for perfusing the organ or tissue via the (one or more) cannulas, and means (e.g., a containment system) for maintaining a sterile environment for the organ or tissue. Cannula insertion and perfusion are techniques well known in the art. The cannula insertion device generally includes a cavity tubing of appropriate size for introducing into the tubes, ducts, and / or cavities of the organ or tissue. Typically, one or more cannulas are inserted into the tubes, ducts, and / or cavities in the organ. The perfusion device may include a support container for a liquid (e.g., a cell disruption medium) and a mechanism (e.g., a pump, pneumatic, gravity) for moving the liquid through the organ via one or more cannulas. The sterility of the organ or tissue during decellularization and / or recellularization can be maintained using various techniques known in the art, such as controlling air flow and filtration, and / or perfusing with, for example, antibiotics, antifungal agents, or other antibacterial agents to prevent the growth of unwanted microorganisms.

[0045] The system for decellularizing and recellularizing the organ or tissue described herein may have the ability to monitor specific perfusion characteristics (e.g., pressure, volume, flow pattern, temperature, gas, pH), mechanical forces (e.g., ventricular wall motion and stress), and electrical stimulation (e.g., pacing). Since the coronary vascular bed (e.g., vascular resistance, volume) changes during the processes of decellularization and recellularization, a pressure-regulated perfusion device is advantageous to avoid significant fluctuations. The effectiveness of perfusion can be evaluated in the effluent and tissue sections. Perfusion volume, flow pattern, temperature, O 2 and CO 2 partial pressures, and pH can be monitored using standard methods.

[0046] Sensors can be used to monitor a system (e.g., a bioreactor), and / or an organ or tissue. Sono-micrometry, micro-manometry, and / or conductance measurements can be used to obtain information on pressure-volume, or preload-recruitable stroke work related to myocardial wall motion and performance. For example, sensors can be used to monitor the pressure of a liquid moving within a cannulated organ or tissue; the environmental temperature in the system and / or the temperature of an organ or tissue; the pH and / or flow rate of a liquid moving within a cannulated organ or tissue; and / or the biological activity of an organ or tissue to be recellularized. In addition to comprising sensors for monitoring such characteristics, a system for decellularizing and / or recellularizing an organ or tissue can also include means for maintaining or regulating these characteristics. Means for maintaining or regulating these characteristics can include components such as thermometers, thermostats, electrodes, pressure sensors, overflow valves, valves for changing the flow rate of a liquid, valves for opening and closing a fluid connection to a solution used to change the pH of the solution, balloons, extracorporeal pacemakers, and / or compliance chambers. Chambers, reservoirs, and pipes may be equipped with cooling devices so that stable conditions (e.g., temperature) can be obtained.

[0047] During recellularization, it may be advantageous to apply mechanical loading to the organ and the cells attached thereto. As an example, a balloon inserted into the left ventricle via the left atrium may be used to apply mechanical stress to the heart. A piston pump that allows adjustment of volume and speed may be connected to the balloon to stimulate left ventricular wall motion and stress. To monitor wall motion and stress, left ventricular wall motion and pressure can be measured using micromanometry, sonomicrometry, pressure-volume changes, or echocardiography. In some embodiments, an external pacemaker can be connected to the piston pump to obtain synchronized stimulation with the contraction of the ventricular balloon (equivalent to systole). Peripheral ECG can be recorded from the heart surface to allow adjustment of the pacing voltage, monitoring of depolarization and repolarization, and obtaining a simplified surface map of the recellularized or recellularizing heart.

[0048] Mechanical ventricular dilation can also be achieved by attaching a peristaltic pump to a cannula inserted into the left ventricle via the left atrium. Similar to the above-described procedure involving a balloon, ventricular dilation obtained with periodic fluid motion (e.g., pulsatile flow) through the cannula can be synchronized with electrical stimulation.

[0049] Using the methods and materials disclosed herein, mammalian hearts can be decellularized and recellularized, and when maintained under appropriate conditions, can give rise to a functional heart that undergoes contractile function and responds to pacing stimuli and / or drugs. This recellularized functional heart can be transplanted into a mammal and function for a period of time.

[0050] Figure 2 shows one aspect of a system (e.g., a bioreactor) for decellularizing and / or recellularizing an organ or tissue. The illustrated aspect is a bioreactor for decellularizing and recellularizing the heart. This aspect includes an adjustable speed and volume peristaltic pump (A); an adjustable speed and volume piston pump (B) connected to an intraventricular balloon; an adjustable voltage, frequency, and amplitude extracorporeal pacemaker (C); an ECG recorder (D); a pressure sensor in the "arterial line" (corresponding to coronary artery pressure) (E); a pressure sensor in the "venous" line (corresponding to coronary sinus ostium pressure) (F); and synchronization (G) between the pacemaker and the piston pump.

[0051] A system for generating an organ or tissue can be controlled by a computer-readable storage medium combined with a programmable processor (e.g., the computer-readable storage medium used herein stores instructions for causing the programmable processor to perform specific steps). For example, such a storage medium combined with a programmable processor can receive and process information from one or more sensors. Such a storage medium used in conjunction with a programmable processor can also send information and instructions back to the bioreactor and / or the organ or tissue.

[0052] An organ or tissue during recellularization can be monitored for biological activity. The biological activity can be that of the organ or tissue itself, such as the electrical activity, mechanical activity, mechanical pressure, contractility, and / or wall stress of the organ or tissue. Furthermore, the biological activity of the cells attached to the organ or tissue can be monitored, for example, for ion transport / exchange activity, cell division, and / or cell viability. See, for example, Laboratory Textbook of Anatomy and Physiology (2001, Wood, Prentice Hall) and Current Protocols in Cell Biology (2001, Bonifacino et al., Eds, John Wiley & Sons). As described above, it may be useful to simulate an active load on the organ during recellularization. The computer-readable storage medium of the present invention can be combined with a programmable processor and used to associate the components necessary to monitor and maintain an active load on an organ or tissue.

[0053] In one aspect, the weight of the organ or tissue can be input into a computer-readable storage medium as described herein and, together with a programmable processor, the exposure time and perfusion pressure for that particular organ or tissue can be calculated. Such a storage medium can record the preload and afterload (the pressures before and after perfusion, respectively), as well as the flow rate. In this aspect, for example, a computer-readable storage medium can, together with a programmable processor, adjust the perfusion pressure, perfusion direction, and / or the type of perfusion solution via one or more pump and / or valve controls.

[0054] According to the present invention, conventional molecular biology, microbiology, biochemistry, and cell biology techniques within the scope of the art may be employed. Such techniques are fully described in the literature. The present invention will be further described in the following examples, which do not limit the scope of the invention as claimed.

Examples

[0055] Section A. Decellularization (Part I) Example 1 - Preparation of Solid Organs for Decellularization To avoid the formation of postmortem thrombi, donor rats were heparinized systemically with 400 U of heparin / kg (donor). After heparinization, the heart and adjacent large blood vessels were completely removed.

[0056] The heart was placed in a physiological saline solution (0.9%) containing heparin (2000 U / ml) and kept at 5 °C until further processing. Under sterile conditions, connective tissue was removed from the heart and large blood vessels. The inferior vena cava and the left and right pulmonary veins were ligated distally from the right and left atria using non-absorbable monofilament ligature thread.

[0057] Example 2 - Cannula Insertion and Perfusion of Solid Organs The heart was placed on a decellularization device for perfusion (Figure 1). A cannula was inserted into the descending thoracic aorta to enable retrograde coronary perfusion (Figure 1, cannula A). Branches of the thoracic aorta (e.g., brachiocephalic trunk, left common carotid artery, left subclavian artery) were ligated. A cannula was inserted into the pulmonary artery just before it divides into the left and right pulmonary arteries (Figure 1, cannula B). A cannula was inserted into the superior vena cava (Figure 1, cannula C). This configuration enables both retrograde and antegrade coronary perfusion.

[0058] When positive pressure was applied to the aortic cannula (A), perfusion occurred from the coronary arteries, through the capillary bed and coronary venous system, to the right atrium and superior vena cava (C). When positive pressure was applied to the superior vena cava cannula (C), perfusion occurred from the right atrium, coronary venous ostium, and coronary veins, through the capillary bed, to the coronary arteries and aortic cannula (A).

[0059] Example 3 - Decellularization After placing the heart on the decellularization device, antegrade perfusion was initiated with cold heparinized calcium-free phosphate buffer containing 1 - 5 mmol of adenosine per liter of perfusate to reconstruct a certain coronary blood flow. Coronary blood flow was evaluated by measuring the coronary perfusion pressure and flow rate and calculating the coronary resistance. After 15 minutes of stable coronary blood flow, a surfactant-based decellularization process was initiated.

[0060] The details of the procedure are described below. Briefly, however, the heart was perfused with surfactant in an antegrade manner. After perfusion, buffer (e.g., PBS) can be flowed retrogradely through the heart. Subsequently, the heart was perfused with PBS containing an antibiotic, and then PBS containing DNase I. Thereafter, the heart was perfused with 1% benzalkonium chloride to reduce microbial contamination and prevent future microbial contamination, and then PBS was perfused to wash away any remaining cellular components, enzymes, or surfactant from the organ.

[0061] Example 4 - Decellularization of Cadaver Rat Hearts Hearts were isolated from 8 male nude rats (250 - 300 g). Immediately after dissection, cannulation was performed on the aortic arch, and the heart was perfused retrogradely with the indicated surfactant. Four different surfactant-based decellularization protocols (see below) were compared for their feasibility and effectiveness in (a) removing cellular components and (b) preserving the vascular structure.

[0062] Decellularization generally included the following steps: stabilization of the solid organ, decellularization of the solid organ, regeneration and / or neutralization of the solid organ, washing of the solid organ, degradation of any DNA remaining on the organ, disinfection of the organ, and organ homeostasis.

[0063] A) Decellularization Protocol #1 (PEG) The heart was washed without recirculation in 200 ml of PBS containing 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 0.25 μg / ml amphotericin B. The heart was then decellularized with 35 ml of polyethylene glycol (PEG; 1 g / ml) manually with recirculation for up to 30 minutes. Next, the organ was washed with 500 ml of PBS using a recirculation pump for up to 24 hours. The washing process was repeated at least twice, each time for at least 24 hours. The heart was exposed to 35 ml of DNase I (70 U / ml) manually with recirculation for at least 1 hour. The organ was washed again with 500 ml of PBS for at least 24 hours.

[0064] B) Decellularization Protocol #2 (TritonX and Trypsin) The heart was washed without recirculation in 200 ml of PBS containing 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 0.25 μg / ml amphotericin B for at least about 20 minutes. The heart was then decellularized by perfusion with 0.05% trypsin for 30 minutes and then with 500 ml of PBS containing 5% Triton-X and 0.1% ammonium hydroxide for about 6 hours. The heart was perfused with deionized water for about 1 hour and then with PBS for 12 hours. Next, using a recirculation pump, the heart was washed three times with 500 ml of PBS, each time for 24 hours. The heart was perfused with 35 ml of DNase I (70 U / ml) manually with recirculation for 1 hour and washed twice in 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump.

[0065] C) Decellularization Protocol #3 (1% SDS) The heart was washed in 200 ml of PBS containing 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 0.25 μg / ml amphotericin B for at least about 20 minutes without recirculation. The heart was decellularized with 500 ml of water containing 1% SDS for at least about 6 hours using a recirculation pump. The heart was then washed with deionized water for about 1 hour and with PBS for about 12 hours. The heart was washed 3 times with 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump. The heart was then perfused with 35 ml of DNase I (70 U / ml) for about 1 hour while manually recirculating and washed 3 times with 500 ml of PBS, each time for at least about 24 hours, using a recirculation pump.

[0066] D) Decellularization Protocol #4 (Triton X) The heart was washed in 200 ml of PBS containing 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 0.25 μg / ml amphotericin B for at least about 20 minutes without recirculation. The heart was then decellularized with 500 ml of water containing 5% Triton X and 0.1% ammonium hydroxide for at least 6 hours using a recirculation pump. The heart was then perfused with deionized water for about 1 hour followed by perfusion with PBS for about 12 hours. The heart was washed 3 times with 500 ml of PBS, each time for at least 24 hours, by recirculation using a recirculation pump. The heart was then perfused with 35 ml of DNase I (70 U / ml) for about 1 hour while manually recirculating and washed 3 times with 500 ml of PBS, each time for about 24 hours.

[0067] For the initial experiments, the decellularization device was placed within a laminar flow hood. The heart was perfused at a coronary perfusion pressure of 60 cm H 2 O. Although not necessary, the hearts described in the above experiments were placed and fully submerged within the decellularization chamber and perfused with PBS containing antibiotics in recirculation mode at a continuous flow rate of 5 ml / min for 72 hours to wash out as many cellular components and surfactants as possible.

[0068] The success of decellularization was defined by the absence of muscle filaments and nuclei in histological sections. The success of the preservation of vascular structures was evaluated by perfusion with 2% Evans blue before embedding the tissue sections.

[0069] The heart was first perfused anterogradely at a constant coronary perfusion pressure with an ionic surfactant (1% sodium dodecyl sulfate (SDS), approximately 0.03 M) dissolved in deionized H 2 2O, and then perfused anterogradely with a nonionic surfactant (1% Triton X-100) to remove SDS and presumably regenerate extracellular matrix (ECM) proteins. Decellularization was very efficient. Intermittently, the heart was perfused retrogradely with phosphate-buffered saline to clear occluded capillaries and small blood vessels.

[0070] Example 5 - Evaluation of Decellularized Organs To demonstrate the intact vascular structures after decellularization, the decellularized heart was stained via Langendorff perfusion with Evans blue to stain the vascular basement membrane and quantify the large and small vessel density. Furthermore, the heart was perfused with polystyrene particles to quantify the coronary volume, i.e., the level of vascular leakage, and the distribution of perfusion could be evaluated by analyzing the coronary effluent and tissue sections. A combination of three criteria was evaluated and compared to isolated non-decellularized hearts: namely, 1) the uniform distribution of polystyrene particles, 2) a significant change in leakage at a certain level, and 3) the small vessel density.

[0071] The fiber orientation was evaluated by the polarization microscopy technique of Tower et al. (2002, Fiber alignment imaging during mechanical testing of soft tissues, Ann Biomed Eng., 30(10):1221-33), which can be applied in real time to samples subjected to uniaxial or biaxial stress. During Langendorff perfusion, the basic mechanical properties (compliance, elasticity, burst pressure) of the decellularized ECM were recorded and compared to freshly isolated hearts.

[0072] Section B. Decellularization (Part II) Example 1 - Decellularization of Rat Hearts Twelve-week-old F344 Fischer male rats (Harlan Labs, PO Box 29176 Indianapolis, IN 46229) were anesthetized using an intraperitoneal injection of 100 mg / kg ketamine (Phoenix Pharmaceutical, Inc., St. Joseph, MO) and 10 mg / kg xylazine (Phoenix Pharmaceutical, Inc., St. Joseph, MO). After systemic heparinization (American Pharmaceutical Partners, Inc., Schaumberg, IL) via the left femoral vein, a median sternotomy was performed and the pericardium was opened. The retrosternal fat body was removed, the ascending thoracic aorta was incised, and its branches were ligated. After disconnecting the vena cava, pulmonary veins, pulmonary artery, and thoracic aorta, the heart was removed from the chest. A pre-filled 1.8 mm aortic cannula (Radnoti Glass, Monrovia, CA) was inserted into the ascending aorta to perform retrograde coronary perfusion (Langendorff). The heart was perfused with heparinized PBS (Hyclone, Logan, UT) containing 10 μM adenosine at a coronary perfusion pressure of 75 cm H 2 O for 15 minutes, followed by perfusion with deionized water containing 1% sodium dodecyl sulfate (SDS) or 1% polyethylene glycol 1000 (PEG 1000) (EMD Biosciences, La Jolla, Germany) or 1% Triton-X 100 (Sigma, St. Louis, MO) for 2 to 15 hours. Thereafter, perfusion with deionized water for 15 minutes and perfusion with deionized water containing 1% Triton-X (Sigma, St. Louis, MO) for 30 minutes were performed. Subsequently, the heart was continuously perfused with antibiotic-containing PBS (100 U / ml penicillin-G (Gibco, Carlsbad, CA), 100 U / ml streptomycin (Gibco, Carlsbad, CA), and 0.25 μg / ml amphotericin B (Sigma, St. Louis, MO)) for 124 hours.

[0073] After 420 minutes of retrograde perfusion with either 1% PEG, 1% Triton-X 100, or 1% SDS, perfusion with PEG and Triton-X 100 induced an edematous and opaque appearance, while SDS perfusion caused more dramatic changes, resulting in a nearly translucent graft as the opaque elements were slowly washed away. Hearts subjected to all three protocols remained extremely intact, with no signs of coronary rupture or aortic valve insufficiency through the perfusion protocol (at a constant coronary perfusion pressure of 77.4 mmHg). Coronary blood flow decreased during the first 60 minutes of perfusion in all three protocols, normalized during SDS perfusion, and remained increased during Triton-X 100 and PEG perfusion. SDS perfusion induced the highest initial increase in calculated coronary resistance (up to 250 mmHg.s.ml -1 ), followed by Triton-X (up to 200 mmHg.s.ml -1 ) and PEG (up to 150 mmHg.s.ml -1 ).

[0074] Using histological sections of surfactant-perfused heart tissue, it was determined that cellularization was incomplete throughout the observation time in both hearts treated with PEG and Triton-X 100. Nuclei and striated filaments were shown by hematoxylin-eosin (HE) staining. In contrast, no nuclei or contractile filaments were detected in sections of SDS-perfused hearts. However, in SDS-treated hearts, the vascular structure and ECM fiber orientation were preserved.

[0075] To remove ionic SDS from the ECM after initial decellularization, the organs were perfused with Triton-X 100 for 30 minutes. Additionally, to ensure complete and thorough washing away of all surfactants and re-establish physiological pH, the decellularized organs were perfused with deionized water and PBS over a wide range for 124 hours.

[0076] Example 2 - Decellularization of Rat Kidneys To isolate the kidneys, the entire peritoneal contents were wrapped in wet gauze and carefully moved laterally to expose the retroperitoneal cavity. The mesenteric vessels were ligated and transected. The abdominal aorta was ligated and transected just below where the renal arteries originated. The thoracic aorta was transected just above the diaphragm, and cannulation was performed using an 1.8 mm aortic cannula (Radnoti Glass, Monrovia, CA). The kidneys were carefully removed from the retroperitoneum and submerged in sterile PBS (Hyclone, Logan, UT) to minimize traction on the renal arteries. After perfusion with heparinized PBS for 15 minutes, perfusion with deionized water containing 1% SDS (Invitrogen, Carlsbad, CA) for 2 - 16 hours, and perfusion with deionized water containing 1% Triton-X (Sigma, St. Louis, MO) for 30 minutes were performed. Subsequently, the liver was continuously perfused with PBS containing antibiotics (100 U / ml penicillin-G (Gibco, Carlsbad, CA), 100 U / ml streptomycin (Gibco, Carlsbad, CA), 0.25 μg / ml amphotericin B (Sigma, St. Louis, MO)) for 124 hours.

[0077] 420 minutes of SDS perfusion followed by Triton-X 100 produced a completely decellularized renal ECM scaffold with an intact vascular system and organ structure. An intact vascular system similar to that of the decellularized heart ECM was confirmed by Evans blue perfusion. Mobat pentachrome staining of the decellularized renal cortex showed intact glomeruli and proximal and distal tubule basement membranes without any intact cells or nuclei. Staining of the decellularized renal medulla showed intact tubules and collecting duct basement membranes. SEM of the decellularized renal cortex confirmed intact glomeruli and tubule basement membranes. Characteristic structures such as the Bowman's capsule delineating the glomeruli from the surrounding proximal and distal tubules and the glomerular capillary basement membrane within the glomeruli were preserved. SEM images of the decellularized renal medulla showed intact medullary pyramids reaching the renal pelvis along with intact collecting duct basement membranes leading to the papilla. That is, all of the major ultrastructures of the kidney were intact after decellularization.

[0078] Example 3 - Decellularization of Rat Lungs (Having a trachea) The lungs were carefully removed from the chest and submerged in sterile PBS (Hyclone, Logan, UT) to minimize traction on the pulmonary artery. After 15 minutes of heparinized PBS perfusion, perfusion with deionized water containing 1% SDS (Invitrogen, Carlsbad, CA) for 2 - 12 hours and perfusion with deionized water containing 1% Triton-X (Sigma, St. Louis, MO) for 15 minutes were performed. Subsequently, the lungs were continuously perfused with antibiotic-containing PBS (100 U / ml penicillin-G (Gibco, Carlsbad, CA), 100 U / ml streptomycin (Gibco, Carlsbad, CA), 0.25 μg / ml amphotericin B (Sigma, St. Louis, MO)) for 124 hours.

[0079] 180 minutes of SDS perfusion followed by Triton-X 100 perfusion produced a completely decellularized lung ECM scaffold with intact airways and vessels. Mobat pentachrome staining of histological sections showed the presence of ECM components in the lungs, including major structural proteins such as collagen and elastin, as well as soluble elements such as proteoglycans. However, no nuclei or intact cells were retained. The airways were preserved from the main bronchi to the terminal bronchioles, respiratory bronchioles, alveolar ducts, and alveoli. The vascular bed from the pulmonary artery to the capillary level and the pulmonary veins remained intact. SEM micrographs of the decellularized lungs showed preservation of the bronchi, alveoli, and vascular basement membranes with no signs of retained cells. The network of elastic and reticular fibers providing major structural support to the alveolar interstitium and septal basement membranes was intact, including the dense network of capillaries in the lung interstitium.

[0080] SEM micrographs of the decellularized trachea showed an intact ECM structure with decellularized hyaline cartilage rings and a rough luminal basement membrane without airway epithelium.

[0081] Example 4 - Decellularization of Rat Livers To isolate the liver, the vena cava was exposed by midline laparotomy, incised, and cannulated using a mouse aortic cannula (Radnoti Glass, Monrovia, CA). The hepatic artery and vein, as well as the bile duct, were transected, and the liver was carefully removed from the abdomen and submerged in sterile PBS (Hyclone, Logan, UT) to minimize traction on the portal vein. After perfusion with heparinized PBS for 15 minutes, perfusion was performed with deionized water containing 1% SDS (Invitrogen, Carlsbad, CA) for 2 - 12 hours and then with deionized water containing 1% Triton-X (Sigma, St. Louis, MO) for 15 minutes. Subsequently, the liver was continuously perfused with PBS containing antibiotics (100 U / ml penicillin-G (Gibco, Carlsbad, CA), 100 U / ml streptomycin (Gibco, Carlsbad, CA), 0.25 μg / ml amphotericin B (Sigma, St. Louis, MO)) for 124 hours.

[0082] A 120-minute SDS perfusion followed by perfusion with Triton-X 100 was sufficient to produce a completely decellularized liver. From the Masson's pentachrome staining of the decellularized liver, it was confirmed that the characteristic liver architecture with central veins and portal vein cavities containing hepatic arteries, bile ducts, and portal veins was retained.

[0083] Example 5 - Methods and Materials Used to Evaluate Decellularized Organs Tissue Structure and Immunofluorescence .Masson's pentachrome staining of paraffin-embedded decellularized tissue was performed according to the manufacturer's instructions (American Mastertech Scientific, Lodi, CA). Briefly, deparaffinized slides were stained using Verhoeff's elastic staining method, rinsed, differentiated in 2% ferric chloride, rinsed, placed in 5% sodium thiosulfate, rinsed, blocked in 3% glacial acetic acid, stained in 1% alcian blue solution, rinsed, stained in crocein scarlet-acid fuchsin, rinsed, immersed in 1% glacial acetic acid, destained in 5% phosphotungstic acid, immersed in 1% glacial acetic acid, dehydrated, placed in alcoholic safranin solution, dehydrated, mounted, and covered with a cover slip.

[0084] Immunofluorescence staining was performed on the decellularized tissue. Antigen retrieval was performed on paraffin-embedded tissue (recellularized tissue) as follows, but not on frozen sections (decellularized tissue): namely, the wax was removed from the paraffin sections, xylene was changed twice for 5 minutes each, and then the sections were subjected to a continuous alcohol gradient, rinsed with cold tap water, and rehydrated. Next, the slides were placed in an antigen retrieval solution (2.94 g trisodium citrate, 22 ml of 0.2 M hydrochloric acid solution, more than 978 ml ultrapure water, adjusted to pH 6.0) and boiled for 30 minutes. After rinsing under cold tap water for 10 minutes, immunostaining was initiated. The frozen sections were fixed with 1×PBS (Mediatech, Herndon, VA) containing 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 15 minutes at room temperature before staining. The slides were blocked with 1×PBS containing 4% fetal bovine serum (FBS; HyClone, Logan, UT) for 30 minutes at room temperature. The samples were continuously incubated with diluted primary and secondary antibodies (Abs) for 1 hour at room temperature. Between each step, the slides were washed three times with 1×PBS (5 - 10 minutes each). The primary antibodies against collagen I (goat polyclonal IgG (Cat. No. sc-8788), Santa Cruz Biotechnology Inc., Santa Cruz, CA), collagen III (goat polyclonal IgG (Cat. No. sc-2405), Santa Cruz Biotechnology Inc., Santa Cruz, CA), fibronectin (goat polyclonal IgG (Cat. No. sc-6953), Santa Cruz Biotechnology Inc., Santa Cruz, CA), and laminin (rabbit polyclonal IgG (Cat. No. sc-20142), Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used at a 1:40 dilution with blocking buffer.The secondary antibodies, goat anti-rabbit IgG phycoerythrin (Cat. No. sc-3747, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and goat anti-mouse IgG phycoerythrin (Cat. No. sc-3750, Santa Cruz Biotechnology Inc., Santa Cruz, CA), were used at a 1:80 dilution in blocking buffer. The slides were covered with coverslips (Fisherbrand 22 x 60, Pittsburgh, PA) in mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector Laboratories, Inc., Burlingame, CA). Images were recorded using ImagePro Plus 4.5.1 (MediaCybernetics, Silver Spring, MD) on a Nikon Eclipse TE200 inverted microscope (Fryer Co. Inc., Huntley, IL).

[0085] Scanning Electron Microscopy. Normal and decellularized tissues were perfusion-fixed with 0.1 M cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA) containing 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) for 15 minutes. The tissues were then rinsed twice in 0.1 M cacodylate buffer for 15 minutes each. Post-fixation was performed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) for 60 minutes. Tissue samples were then dehydrated in ascending concentrations of EtOH (50% for 10 minutes, 70% for 10 minutes twice, 80% for 10 minutes, 95% for 10 minutes twice, 100% for 10 minutes twice). The tissue samples were then subjected to critical point drying in a Tousimis Samdri-780A (Tousimis, Rockville, MD). Coating was performed by gold / palladium sputter coating for 30 seconds in a Denton DV-502A vacuum evaporator (Denton Vacuum, Moorestown, NJ). Scanning electron microscope images were taken using a Hitachi S4700 field emission scanning electron microscope (Hitachi High Technologies America, Pleasanton, CA).

[0086] Mechanical Testing. Crosses of myocardial tissue were excised from the left ventricle of rats such that the central region was approximately 5 mm × 5 mm and the axes of the crosses were oriented along the periphery and longitudinal direction of the heart. When the initial thickness of the tissue crosses was measured with a micrometer, it was 3.59 ± 0.14 mm at the center of the tissue crosses. Crosses were also excised from decellularized rat left ventricular tissue with the same orientation and the same central region size. The initial thickness of the decellularized samples was 238.5 ± 38.9 μm. Furthermore, the mechanical properties of fibrin gels were examined and another tissue engineering scaffold was used to engineer blood vessels and heart tissue. Fibrin gels were poured into a cross-shaped mold at a final concentration of 6.6 mg of fibrin / ml. The average thickness of the fibrin gels was 165.2 ± 67.3 μm. All samples were attached via clamps to a biaxial mechanical testing machine (Instron Corporation, Norwood, MA), submerged in PBS, and pulled equibiaxially at 40% strain. To accurately examine the static and passive mechanical properties, the samples were pulled in 4% strain increments and relaxed for at least 60 seconds at each strain value. The force was converted to engineering stress by normalizing the force value with respect to the cross-sectional area in a specific axial direction (5 mm × initial thickness). The engineering stress was calculated as the displacement normalized by the initial length. To compare the data between the two axes and between sample groups, the tangent modulus was calculated as follows: [T(ε = 40% strain) - T(ε = 36% strain)] / 4% strain where T is the engineering stress and ε is the engineering strain. The values of the tangent modulus were averaged and compared between the two axes (periphery and longitudinal) and between groups.

[0087] Example 6 - Evaluation of the Biocompatibility of Decellularized Organs To evaluate biocompatibility, 100,000 mouse embryonic stem cells (mESCs) suspended in 1 cc of standard expansion medium (Iscove's Modified Dulbecco's Medium (Gibco, Carlsbad, CA), 10% fetal bovine serum (HyClone, Logan, UT), 100 U / ml penicillin-G (Gibco, Carlsbad, CA), 100 U / ml streptomycin (Gibco, Carlsbad, CA), 2 mmol / L L-glutamine (Invitrogen, Carlsbad, CA), 0.1 mmol / L 2-mercaptoethanol (Gibco, Carlsbad, CA)) were seeded onto ECM sections and control plates without special growth factor stimulation or feeder cell support. 4',6-Diamidino-2-phenylindole (DAPI) was added to the cell culture medium at a concentration of 10 μg / ml to label cell nuclei and enable quantification of cell attachment and spreading. Images under UV light, as well as phase contrast at reference points, 24, 48, and 72 hours later, were recorded using ImagePro Plus 4.5.1 (Media Cybernetics, Silver Spring, MD) on a Nikon Eclipse TE200 inverted microscope (Fryer Co. Inc., Huntley, IL).

[0088] The decellularized ECM was compatible with cell viability, attachment, and proliferation. The seeded mESCs adhered to the ECM scaffolds and began to invade the matrix within 72 hours of cell seeding.

[0089] Example 7 - Evaluation of Decellularized Organs The aortic valve competency and the integrity of the coronary vascular bed of the SDS-decellularized rat heart were evaluated by Langendorff perfusion with 2% Evans blue dye. Since no left ventricular filling with the dye was observed, it was shown that the aortic valve was intact. Macroscopically, filling up to the fourth branch point of the coronary artery was confirmed without signs of dye leakage. Subsequently, in tissue sections, perfusion of large (150 μm) and small (20 μm) arteries and veins was confirmed by red fluorescence of the Evans blue-stained vascular basement membrane.

[0090] To confirm the retention of major cardiac ECM components, immunofluorescent staining of SDS decellularized ECM scaffolds was performed. This confirmed the presence of major cardiac ECM components such as collagen I and III, fibronectin, and laminin, but there were no signs of intact nuclei or contractile elements containing cardiac myosin heavy chain or sarcomeric alpha-actin being retained.

[0091] Scanning electron micrographs (SEM) of SDS decellularized cardiac ECM demonstrated that fiber orientation and composition were preserved in the aortic wall and aortic valve leaflets where cells were absent throughout the tissue thickness. The decellularized left and right ventricular walls retained the ECM fiber composition (weave, strut, coil) and orientation, and the muscle fibers were completely removed. In the retained ECM of both ventricles, intact vascular basal laminae of different diameters without endothelial or smooth muscle cells were observed. Additionally, a thin layer of epicardial fibers with high density was retained under the intact epicardial basement membrane.

[0092] To evaluate the mechanical properties of decellularized cardiac tissue, biaxial testing was performed and compared to fibrin gels, which are commonly used as artificial ECM scaffolds in cardiac tissue engineering. Normal rat ventricles and decellularized samples were highly anisotropic with respect to stress-strain behavior. In contrast, in fibrin gel samples, the stress-strain properties were very similar between the two principal directions. Direction-dependence of stress-strain behavior was present in all samples of the normal rat ventricle and decellularized groups, and isotropy of stress-strain properties was unique to all samples of the fibrin gel group.

[0093] To compare the stress-strain properties between these two groups and between the major axes of the heart, the tangent modulus was calculated at 40% strain in both the circumferential and longitudinal directions (see Example 5 for the equations). Note that in both directions, the decellularized sample group had a significantly higher modulus than the normal rat ventricle and fibrin gel sample groups. However, there was a significant difference in the modulus between the two directions for both the normal rat ventricle and the decellularized matrix, but not for the fibrin gel.

[0094] For intact left ventricular tissue, the stress at 40% strain varied from 5 - 14 kPa in the longitudinal direction and from 15 - 24 kPa in the circumferential direction, which is consistent with previously published data. In both rat ventricular tissue and decellularized rat ventricular tissue, the circumferential direction was stiffer than the longitudinal direction, most likely due to the orientation of the cardiac muscle fibers. Although the fiber orientation varies through the thickness of the cardiac tissue, most of the fibers are oriented circumferentially, so it is expected to be stiffer along this direction. The decellularized tissue was significantly stiffer than the intact tissue. This can also be expected since the extracellular matrix is stiffer than the cells themselves and the combination of ECM and cells is not as stiff as the ECM alone. The values of the tangent modulus for the decellularized tissue seem to be relatively large, but are only slightly larger than the value of the Young's modulus for purified elastin (approximately 600 kPa) and smaller than the Young's modulus for a single collagen fiber (5 Mpa), and the values determined herein are within a reasonable range.

[0095] Example 8 - Decellularization of Other Organs or Tissues Similar results were obtained when the perfusion-based decellularization protocol described herein was applied to skeletal muscle, pancreas, small intestine, large intestine, esophagus, stomach, spleen, brain, spinal cord, and bone in addition to rat heart, lung, kidney, and liver.

[0096] Example 9 - Decellularization of Porcine Kidneys Porcine kidneys were isolated from heparinized male animals. To perfuse the isolated organs, cannulas were inserted into the renal arteries and blood was washed out with PBS perfusion for 15 minutes. Perfusion with 27 L of deionized water containing 1% SDS was carried out for 35.5 hours under a pressure of 50 - 100 mmHg. Perfusion with deionized water containing 1% Triton-X-100 was initiated to remove SDS from the ECM scaffold. Subsequently, the decellularized kidneys were washed and buffered by perfusion with PBS containing antibiotics for 120 hours to remove the surfactant and obtain a biocompatible pH.

[0097] Cleaning of the organs was observed within 2 hours from the start of perfusion. A clean white color appeared 12 hours after perfusion. Decellularization was terminated when the organs became translucent white.

[0098] Example 10 - Transplantation of Decellularized Hearts Hearts from F344 rats were prepared by cannulating the aorta distal to the aortic valve and ligating all other major and pulmonary vessels except the left branch of the pulmonary trunk (distal to the bifurcation) and the inferior vena cava (IVC). Decellularization was achieved by Langendorff retrograde coronary perfusion and using 2 L of 1% SDS over 12 - 16 hours. The hearts were then repopulated with 35 mL of 1% Triton-X-100 over 30 - 40 minutes and washed with PBS containing antibiotics and antifungals for 72 hours. The IVC was ligated before transplantation.

[0099] Large (380 - 400 gram) RNU rats were prepared to receive the decellularized hearts. A blunt-tipped mosquito clamp was applied to both the IVC and the abdominal aorta of the host animal to ensure isolation of the anastomotic area. The aorta of the decellularized heart was anastomosed to the host abdominal aorta proximal and inferior to the renal branch using 8 - 0 silk sutures. The left branch of the pulmonary trunk of the decellularized heart was anastomosed to the area of the host IVC closest to minimize physical stress on the pulmonary trunk.

[0100] After suturing both tubes into the host animal, the clamp was released and the decellularized heart was filled with the host animal's blood. In the decellularized heart and aorta, the abdominal aortic pressure of the recipient animal was visually observed. The decellularized heart dilated and became red with blood. Bleeding at the anastomosis site was minimal. Heparin was administered 3 minutes after releasing the clamp (initiation of perfusion), the heart was photographed, placed in the abdomen to minimize stress on the anastomosis site. The abdomen was closed aseptically and the animals were monitored for recovery. 55 hours after transplantation, the animals were euthanized and the decellularized hearts were explanted for observation. Animals that did not receive heparin showed large thrombi in the LV during incision and evaluation. Also, blood was observed in the coronary arteries on both the left and right sides of the heart.

[0101] In other transplantation experiments, after suturing both tubes into the host animal, the clamp was released and the decellularized heart was filled with the host animal's blood. In the decellularized heart and aorta, the abdominal aortic pressure of the recipient animal was visually observed. The decellularized heart dilated, became red, and bleeding at the anastomosis site was minimal. Heparin was administered by IP injection (3000 IU) 3 minutes after releasing the clamp (initiation of perfusion). The heart was photographed, placed in the abdomen to minimize stress on the anastomosis site. The abdomen was closed aseptically and the animals were monitored for recovery. Approximately 48 hours after transplantation, the animals were found to have died due to bleeding. Currently, the transplantation time ranges from 55 to 70 minutes.

[0102] Section C. Recellularization Example 1 - Recellularization of Heart ECM Slices To evaluate the biocompatibility of the decellularized ECM, 1-mm-thick slices of one decellularized heart were cultured with myogenic and endothelial cell lines. 2×10 5Rat skeletal myoblasts, C2C12 mouse myoblasts, human umbilical vein endothelial cells (HUVECs), and bovine pulmonary endothelial cells (BPECs) were seeded onto tissue sections and co-cultured for 7 days under standard conditions. Myoblasts migrated and expanded within the ECM and aligned with the original fiber orientation. These myoblasts showed increased proliferation and completely reassembled in most of the ECM laminae. The endothelial cell line showed a pattern of less wet growth and formed a monolayer on the graft surface. Under these conditions, no detectable anti-proliferative effect was observed.

[0103] Example 2 - Recellularization of Heart ECM by Coronary Perfusion To determine the efficiency of seeding regenerative cells onto and into the decellularized heart ECM by coronary perfusion, the decellularized heart was transferred to an organ chamber and continuously perfused with oxygenated cell culture medium under cell culture conditions (5% CO 2 , 60% humidity, 37 °C). 120 × 10 6 PKH-labeled HUVECs (suspended in 50 ml of endothelial cell growth medium) were injected at a coronary perfusion pressure of 40 cm H 2 O. Coronary effluent was collected and the cells were counted. The effluent was then recirculated and perfused again to deliver the maximum number of cells. The recirculation was repeated twice. After the third passage, approximately 90 × 10 6 cells were retained within the heart. The heart was continuously perfused for 120 hours with 500 ml of recirculating oxygenated endothelial cell culture medium. The heart was then removed and embedded for cryosectioning. HUVECs were restricted to the arterial and venous residues throughout the heart but were not yet completely dispersed into the extravascular ECM.

[0104] Example 3 - Recellularization of Decellularized Rat Hearts Using Neonatal Rat Heart Cells Isolation and Preparation of Neonatal Rat Heart Cells. On the first day, 8 - 10 1 - 3 - day - old SPF Fisher - 344 neonatal rats (Harlan Labs, Indianapolis, IN) were anesthetized with 5% inhaled isoflurane (Abbott Laboratories, North Chicago, IL), sprayed with 70% EtOH, and immediately a median sternotomy was performed aseptically. The heart was excised and immediately placed into a 50 - ml conical tube on ice containing HBSS (Reagent #1 from neonatal cardiomyocyte isolation system, Worthington Biochemical Corporation, Lakewood, NJ). The supernatant was removed, and the whole heart was washed once with cold HBSS by vigorous swirling. The heart was transferred to a 100 - mm culture dish containing 5 ml of cold HBSS, connective tissue was removed, and the remaining tissue was minced (<1 mm 2 ). Additional HBSS was added to bring the total plate volume to 9 ml, and 1 ml of trypsin (Reagent #2, Worthington kit) was added to this to obtain a final concentration of 50 μg / ml. The plate was incubated overnight in a 5°C cooler.

[0105] On the second day, the plate was removed from the cooler and placed in a sterile hood on ice. The tissue and trypsin - containing buffer were transferred to a 50 - ml conical tube on ice using a wide - mouth pipette. Trypsin inhibitor (Reagent #3) was reconstituted with 1 ml of HBSS (Reagent #1), added to the 50 - ml conical tube, and gently mixed. Air was blown over the surface of the liquid, and the tissue was oxygenated for 60 - 90 seconds. Then the tissue was warmed to 37°C, and collagenase (300 units / ml) reconstituted with 5 ml of Leibovitz L - 15 was slowly added. The tissue was placed in a warm (37°C) stirrer bath for 45 minutes. Next, the tissue was titrated 10 times using a 10 - ml pipette to release the cells (3 ml / sec), and then filtered through a 0.22 - μm filter. The tissue was washed with an additional 5 ml of L - 15 medium, a second titration was performed, and the cells were collected into the same 50 - ml conical tube. Then, the cell solution was incubated at room temperature for 20 minutes and centrifuged at 50×g for 5 minutes to pellet the cells. The supernatant was gently removed, and the cells were resuspended using neonatal cardiomyocyte medium to the desired volume.

[0106] Media and Solutions . All media were sterilized by filtration and stored in the dark at 5 °C in a cooler. The Worthington isolation kit contains Leibovitz L-15, a recommended culture medium. This medium was used only for tissue processing on the second day. For plating, the alternative calcium-containing medium described herein was used. Worthington Leibovitz L-15 medium: Leibovitz medium powder was reconstituted using 1 L of cell culture grade water. Leibovitz L-15 medium contains 140 mg / ml CaCl, 93.68 mg / ml MgCl, and 97.67 mg / ml MgS. Neonatal cardiomyocyte medium: Iscove's modified Dulbecco's medium (Gibco, Cat. No. 12440-053) was supplemented with 10% fetal bovine serum (HyClone), 100 U / ml penicillin-G (Gibco), 100 U / ml streptomycin (Gibco), 2 mmol / L L-glutamine (Invitrogen), and 0.1 mmol / L 2-mercaptoethanol (Gibco, Cat. No. 21985-023), and treated with a sterile filter before use. Amphotericin-B was added as needed (final concentration 0.25 μg / ml). This medium was fortified with 1.2 mM CaCl (Fisher Scientific, Cat. No. C614-500) and 0.8 mM MgCl (Sigma, Cat. No. M-0250).

[0107] In Vitro Culture Analysis of Recellularization . As a step for fabricating a bioartificial heart, the isolated ECM was recellularized with neonatal heart-derived cells. A completely decellularized heart (fabricated as described herein) was injected with a combination of 50 × 10 6 freshly isolated rat neonatal cardiomyocytes, fibroblasts, endothelial, and smooth muscle cells. The heart tissue was then sliced into thin sections, and the thin sections were cultured in vitro to examine the biocompatibility of the decellularized ECM and the ability of the resulting construct to develop into a myocardium ring.

[0108] When the minimum shrinkage in the obtained ring was observed microscopically after 24 hours, it was demonstrated that the transplanted cells could adhere to and engraft in the decellularized ECM. Under the microscope, the cells were oriented along the direction of the ECM fibers. Immunofluorescence staining confirmed the survival and engraftment of cardiomyocytes expressing cardiac myosin heavy chain. Within 4 days, clusters of contracting cell patches were observed on the decellularized matrix, which developed into tissue rings that contracted synchronously on the 8th day.

[0109] On the 10th day, these rings were placed between two rods and the contractile force was measured under different preload conditions. The rings could be electrically paced up to a frequency of 4 Hz maximum and generated a maximum contractile force of 3 mN at a preload of 0.65 g maximum. Thus, according to this in vitro tissue culture approach of recellularization, a contractile tissue was obtained that generated an effective force equivalent to that generated by an optimally engineered and manipulated cardiac tissue ring using an artificial ECM construct.

[0110] Recellularization of Decellularized Hearts via Perfusion . Recellularization (50×10 6 freshly isolated neonatal rat cardiomyocytes, fibroblasts, endothelial and smooth muscle cells) scaffolds were placed in a perfusable bioreactor (n = 10) that mimicked rat heart physiology, including pulsatile left ventricular dilation with gradually increasing preload and afterload (day 1: preload 4 - 12 mmHg, afterload 3 - 7 mmHg), pulsatile coronary blood flow (day 1: 7 ml / min), and electrical stimulation (day 2: 1 Hz) under sterile cardiac tissue culture conditions (5% CO 2 , 60% H 2 O, 37 °C). The perfused organ cultures were maintained for 1 - 4 weeks. Pressure, flow, and EKG were recorded every 15 minutes for 30 seconds throughout the culture period. Videos of the developing bioartificial heart were recorded on days 4, 6, and 10 after cell seeding.

[0111] Ten days after cell seeding, a pressure probe was inserted into the left ventricle to record left ventricular pressure (LVP), and wall motion was video-recorded while gradually increasing the stimulation frequency from 0.1 Hz to 10 Hz, and a more thorough functional evaluation was performed, including pharmacological stimulation with phenylephrine (PE). The recellularized heart showed a contractile response to single pace, showed spontaneous contractions after paced contractions, and the corresponding LVP increased. After single pacing, the heart showed three spontaneous contractions and turned into a fibrillation state. Similar to the facilitated contractions, spontaneous depolarization resulted in a corresponding increase in LVP and a recordable QRS complex, presumably indicating the formation of a developing stable conduction pattern.

[0112] Once the stimulation frequency was increased up to 0.4 Hz, on average two spontaneous contractions occurred after the induced contractions. At a pacing frequency of up to 1 Hz, only one spontaneous contraction occurred. At a pacing frequency of 5 Hz, no spontaneous contractions occurred. The maximum capture rate was 5 Hz, which is consistent with the 250 ms refractory period for mature myocardium. After perfusion with 100 μM PE, normal spontaneous depolarization occurred at a frequency of 1.7 Hz, associated with the corresponding increase in LVP.

[0113] On histological analysis on day 10, cell dispersion and engraftment were revealed throughout the thickness of the left ventricular wall (0.5 - 1.2 mm). Cardiomyocytes aligned along the ventricular fiber direction, forming regions of grafts organized at a high density similar to mature myocardium and regions of immature grafts with a lower density similar to developing myocardium. The cardiomyocyte phenotype was confirmed by immunofluorescent staining for cardiac myosin heavy chain. A high capillary density was maintained across the newly developed myocardium, and the average distance between capillaries was approximately 20 μm, which was similar to that reported for mature rat myocardium. The endothelial cell phenotype was confirmed by immunofluorescent staining for von Willebrand factor (vWF). Cell viability was maintained throughout the graft thickness, indicating sufficient oxygen and nutrient supply through coronary perfusion.

[0114] Section D. Additional Decellularization and Recellularization Example 1 - Rat Liver Isolation Procedure Each rat was anesthetized with 75 mg of ketamine per kg of body weight and 10 mg of xylazine per kg of body weight. The abdomen of the rat was shaved and sterilized with Betadine. A large dose of sodium heparin (100 μL of heparin (1,000 UI / mL stock) per 100 g of body weight) was intravenously administered to the rat via the intragastric vein.

[0115] While the heparin was taking effect, the bioreactor flask was assembled. Briefly, a Tygon tube was attached to a 250 mL flask (connected to the side of the base), and a reducing pipe adapter was attached to the pipe (which acts as a drain during the cleaning process described below). While the heparin was taking effect, a catheter with a rubber stopper was assembled; that is, a 12 cc syringe was filled with PBS, and a three-way stopcock was attached to the syringe. An 18-gauge needle was attached to the syringe and pushed through a No. 8 rubber stopper. It is preferable to keep the needle flat against the bottom of the stopper so that the liver lies flat in the tube. A short polyethylene tube with a melt flange (e.g., PE160) was slipped over the free end of the tube after alcohol sterilization. A small amount of PBS was pushed through the catheter to flush out the alcohol, and a 10 cm Petri dish was filled with enough PBS to cover the isolated liver.

[0116] After circulating the heparin, the abdominal skin was incised to expose the underlying abdominal muscles. After performing a mid-laparotomy, a lateral transverse incision or a midline incision along the abdominal wall was made, and the liver was pulled out and exposed. The ligaments attaching the liver to the duodenum, stomach, diaphragm, and anterior abdominal wall were gently cut (the Glisson's capsule is fragile). The common bile duct, hepatic artery, and portal vein were cut, leaving enough length to insert a catheter, and finally, the inferior vena cava above the liver was severed. The liver was removed while firmly grasping the remaining attached inferior vena cava on the liver and placed in a Petri dish containing PBS. All remaining ligaments were excised.

[0117] Example 2 - Decellularization of the Liver The prepared catheter was inserted into the portal vein and ligated with proline suture. The integrity of the line was verified, and perfusion using PBS (without Mg +2 and Ca +2 contained) in a syringe was used to remove occult blood from the liver. A liver rubber stopper was fitted into the bioreactor. The flask was placed on the collection reservoir, and a container of 1% SDS (1.6 L) was attached via a line of sufficient length to create a column that generated a maximum pressure of approximately 20 mm Hg. After 2 - 4 hours of perfusion, the 1% SDS container was emptied and refilled with an additional 1.6 L of 1% SDS. A total of four 1.6 L batches of 1% SDS were typically used to perfuse the liver. After decellularization, the liver appeared as a clean white color and the vasculature was visible.

[0118] On the second day, the SDS reservoir was removed and replaced with a 60 ml syringe filled with dH 2 O. After rinsing with water, 60 mL of 1% Triton X - 100 was followed by 60 mL of a wash solution containing dH 2 O. The rinsed liver was placed for washing, and perfusion was initiated with PBS containing an antibacterial agent (e.g., penicillin - streptomycin (e.g., Pen - Strep®)) using a small pump (Masterflex, 50% maximum capacity at approximately 1.5 mL / min). A Tygon tube was extended from the bioreactor / flask outlet to the PBS reservoir. The tubing was extended from the pump to a 0.8 micron filter attached to a three - way stopcock on the flask. An 18 - gauge injection needle was attached to the tubing inside the PBS reservoir and kept lower than the introduction line. After 6 hours, the wash solution was replaced with fresh PBS w / Pen - Strep at 1× concentration, the 0.8 micron filter was replaced, and the organ was washed overnight.

[0119] On the third day, washing was continued by further replacing the 500 ml of 1× strength PBS w / Pen-Strep twice. The 0.8 micron filter was replaced each time the PBS was changed. The third wash started in the morning, was changed after 6 hours, and the final wash was continued overnight again. On the fourth day, the liver was ready for recellularization.

[0120] The liver washed twice with 1.6 L of 1% SDS retained an average of 14.27% DNA, and the liver washed four times with 1.6 L of 1% SDS retained an average of 5.36% DNA. That is, two washes with 1% SDS removed approximately 86% of the DNA (compared to the cadaver), and four washes with 1% SDS removed approximately 95% of the DNA (compared to the cadaver).

[0121] Figure 3A shows the decellularization of rat liver and rat kidney, and Figure 3B shows the decellularization of rat heart and rat lung. The middle part contains a photo during the progress of decellularization, and the left and right photos are SEM images of the decellularized organs. Figure 4 shows the decellularized porcine kidney and rat kidney being perfused with dye, and also shows EM photos of the glomeruli and tubules of the decellularized kidney. Figure 5 shows the whole decellularized rat cadaver as described herein.

[0122] Example 3 - Recellularization of the Liver Recellularization was performed by suspending cells (40 million primary liver-derived cells or HepG2 human cells) in warmed medium (37°C) at approximately 8 million cells per milliliter (typically 5 mL) and loading them into a syringe needle. The cells were injected via the portal vein, during which time the liver was placed inside a bioreactor or in a Petri dish. It should be noted that additionally or alternatively, the cells can be injected via any other vascular access or be injected substantially directly.

[0123] Primary liver-derived cells were obtained by enzymatic digestion of adult rat livers using the Worthington enzyme dissociation kit. Briefly, prior to removal from the rat, the rat liver was perfused via the portal vein with 1× Hank's balanced salt solution without calcium and magnesium (kit vial #1) at 20 mL / min for 10 minutes. Next, the liver was recirculated with 100 mL of L-15 containing the MOPS buffer-containing enzymes (collagenase (22,500 units), elastase (30 units), and DNase I (1,000 units)) obtained from kit vials #2 and #3 at 20 mL / min for 10 - 15 minutes. Thereafter, mechanical disruption of the organ was performed to release the cells. Prior to use for recellularization, the cells were centrifuged at 100 g and resuspended twice in the culture medium.

[0124] Based on visual cues observed during the process (e.g., the tension of the perfused liver lobe, cell escape from the liver, and cell distribution across the target liver lobe), the perfusion rate was controlled. After recellularization, the liver in the bioreactor was placed in an incubator at 37 °C and 5% CO 2 An oxygenated medium reservoir (containing 50 mL of medium) was attached, and the medium in the reservoir was bubbled with humidified carbogen (95% oxygen, 5% carbon dioxide). Using a peristaltic pump, the medium (37 °C) was recirculated through the liver at a rate in the range of 2 - 10 mL / min. The medium was exchanged daily for 7 days to maintain the recellularized rat liver (however, the experiment was terminated when convenient). The medium samples were stored at -20 °C during daily exchanges and assayed for albumin and urea. On day 7, a cytochrome P-450 assay was performed.

[0125] Figure 6 shows the recellularization of a decellularized rat liver. A hypodermic needle was inserted into a single lobe via the portal vein catheter, and primary hepatocytes were injected. Figure 7 shows the targeted delivery of primary rat hepatocytes to the caudate lobe (A) or the lower / upper right lobe (B) of a decellularized rat liver.

[0126] Figure 8 shows scanning electron microscope photographs (SEM) of the recellularized rat liver cultured for one week. These data show the similarity between the cadaveric liver and the recellularized liver at the microstructural level. The cells were incorporated into the matrix bed and had a similar shape to those in freshly isolated cadaveric tissue. Figure 9 shows Masson's trichrome (A) and HE (B) staining, Figure 10A shows TUNEL analysis, and Figure 10B shows Masson's trichrome staining of the recellularized rat liver one week after injecting rat hepatocytes into the caudate process. These results demonstrate that hepatocytes can be delivered, retained in the matrix, and maintained viable by nutrient perfusion.

[0127] Figure 11 shows Masson's trichrome staining of the recellularized rat liver one week after injecting the human hepatocyte line (HepG2) into the caudate process (A) or the upper / lower right lobe (B). Figure 12 is a graph showing cell retention of primary rat hepatocytes (1 - 6) and the human HepG2 cell line (7 and 8). The total number of cells perfused into the liver was counted before injection, and the non - adherent cells that passed through the matrix and finally flowed into the Petri dish were counted. This difference represents the cells retained in the matrix. Figure 13 is a graph showing that human HepG2 cells are viable and proliferate after injection into the decellularized rat liver.

[0128] Example 4 - Liver Function The functions of the decellularized and recellularized livers were evaluated as follows. Urea production (Figure 14), albumin production (Figure 15), and cytochrome P-450IAI (ethoxyresorufin-O-deethylase (EROD)) activity (Figure 16) in the livers recellularized with primary rat hepatocytes were evaluated. Urea production was determined using a Berthelot / colorimetric assay kit (Pointe Scientific Inc.), and albumin production and EROD activity were assayed using methods adapted from Culture of Cells for Tissue Engineering (Vunjak-Novakovic & Freshney, eds., 2006, Wiley-Liss). These experiments demonstrated that liver-derived cells retained liver-specific functionality during the culture period.

[0129] Example 5 - Cell Viability After Recellularization Figure 17 is a graph showing that fetal and adult-derived stem / progenitor cells proliferated on decellularized hearts, lungs, livers, and kidneys for at least 3 weeks. Cell proliferation was determined by counting the number of nuclear DAPI stains per high-power field. Figure 18 is a graph showing that mouse embryonic stem cells (mESCs) and proliferating adult muscle progenitor cells (skeletal myoblasts; SKMB) were viable on decellularized hearts, lungs, livers, and kidneys. Cell viability was determined by detecting the extent of apoptosis after 3 weeks, versus the total number of DAPI-stained cell nuclei, using a tunnel assay.

[0130] Human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells proliferated on the decellularized heart matrix for at least one week. Briefly, human ES cells (H9 obtained from WiCell Research Institute; WA09 obtained from the National Stem Cell Bank (NSCB)) and IMR90 subclones of human iPS cells (generated using OCT4, SOX2, NANOG, and LIN28 lentiviral transgenes as described in Zhang et al., 2009, Circ. Res., 104:e30-e41 and obtained from Dr. Timothy Kamp at the University of Wisconsin) were compared on the decellularized matrix. H9 cells and iPS cells, which contained 20 - 50% cardiac cells among the proliferating fibroblasts, along with other non-beating cells, were plated at densities of 200,000 cells and 90,000 cells, respectively, into wells containing the chamber-specific (right or left atrium or ventricle) portions of the rat decellularized heart matrix that had been isolated and exposed to the interior of the matrix. The cells were simply deposited on the decellularized matrix. The cells were grown in medium containing 20% serum for 3 days and then the serum was lowered to 2% for the next 4 days, in line with the in vitro "shift" of the proliferating muscle cells to a beating muscle cell phenotype. Control cells were plated into the same wells coated with gelatin (0.1%) and grown under the same conditions. The cells were grown in EB20 medium. The cultures were evaluated daily by microscopy and the beating cells were recorded with a video camera. After one week of culture, a live / dead assay was performed to examine cell viability. Additionally, immunohistochemistry was performed to demonstrate the presence of heart-related proteins. It was observed that the cells growing on the decellularized matrix started beating on the 3rd - 4th day, while the cells on gelatin did not beat. On the 5th day, the cells on the matrix expanded and cells beating in a larger area were observed. On the cells grown on gelatin under the same conditions, the beating was either minimal or absent.

[0131] Example 6 - Recellularization Process Cell Isolation Using the Washington protocol, the LV and RV from neonatal rats were isolated by cutting through the approximate center of the heart. The area from the base to the second LAD branch was discarded, and the remainder was placed in approximately 10 mL of HBSS. Optionally, the LV and RV portions of the heart may be incubated overnight at 5 °C in trypsin for up to 18 - 22 hours. After drawing the cells into a syringe for injection into the decellularized matrix, the remaining cells were used as a control (e.g., 10 mL of medium was added and the cells were plated).

[0132] Extracellular Matrix Well - washed decellularized extracellular matrix (ECM) was obtained. For example, the heart extracellular matrix was washed for 3 - 4 days with at least 2000 mL of PBS solution. A 18 ga. cannula (IN: mitral valve through the LV; OUT: aorta) was inserted into the heart and fixed using 4 - 0 suture. The LV cannula was advanced to near the tip within the LV lumen (e.g., the tip of the LV cannula was approximately 0.7 cm from the mitral valve). The structure was checked for any leaks. Optionally, a "high - speed" test can be performed by starting the pump in the [pre - heart] flow probe range of 25 - 28 mL / min for at least 5 - 10 seconds to ensure a secure ECM connection before introducing the cells.

[0133] Cell Injection It was placed in a 100 - 120 mL media bioreactor, and a 60 mm culture plate was placed under the tip of the heart to receive the excess cells, avoid coronary occlusion, and avoid apoptosis signaling from mutant cells. The cells were injected using a 27 ga needle and a 1 cc TB syringe. Approximately 70 μL of cells were injected into the ventricular wall per injection, and the needle entry angle was 15 degrees relative to the normal. The cells were injected 10 - 12 times into the anterior LV wall and 3 - 4 times into the tip of the heart. The total volume of the injected cells should be approximately 1.3 - 1.5 mL. Some backflow and cell loss are expected. The heart was lowered into the bioreactor, and the pump and tank (95% O 2and 5% CO 2 ) was activated, and the heart was monitored for leakage, flow problems, and any other technical problems. The next day, the reactor was opened and the pace line was attached. Frequency: 1 Hz; Delay: 170 MS; Duration: 6 MS; Voltage range: 45 - 60 V; Flow rate (IN): 18 - 22 mL / min; Flow rate (OUT): 14 - 18 mL / min; Difference, about 6 - 7 mL / min, and pacing (continuous) was started.

[0134] Media The following recipe is for 1 liter. To IMDM, add 100 mL FBS 10%; 5 mL Pen Strep; 10 mL L-Glut; 168 μL Amp-B; 1 mL B-Mercap; 20 mL horse serum; 180 mg Ca 2+ ; 96 mg Mg 2+ ; and 50 mg vitamin C.

[0135] NNCM (NEO) Cells Neonatal cardiomyocytes (NNCM or NEO cells) were obtained from the Worthington kit prep. The NEO cells were temperature sensitive. When they dropped to about 35°C, they stopped beating in the same way. The NEO cells, although not confluent, started beating on the 2D plate within 24 hours. When the NEO cells grew and started beating together, they grew on top of each other and started beating synchronously. Eventually, the cells mechanically restricted themselves and usually stopped beating on days 10 - 16.

[0136] Example 7 - Structural Comparison of Decellularized and Recellularized Organs in Cadaver Organs Figure 19 is a SEM photograph of a decellularized heart (right panel) and a cadaveric heart (left panel). The SEM photographs were obtained for both the left ventricle (LV) and the right ventricle (RV). As can be seen from the photographs, the perfusion decellularized heart lacks cellular components but retains the intact myocardial spatial and structural features, including the vasculature. Furthermore, in the perfusion decellularized matrix, it can be seen that structural features including weave (w), coil (c), and cross-strand (s) are retained even though the cells have been completely lost.

[0137] Figure 20 shows a histological (upper) and SEM (lower) comparison of a decellularized and recellularized rat liver (right panel) as described herein, compared to a cadaveric rat liver (left panel). These results show the morphological similarity and structural organization between healthy hepatocytes from an intact liver and hepatocytes cultured or seeded on the decellularized liver. The HE images show that the cells within the recellularized liver began to radially organize around the vasculature, similar to the structure seen in a freshly isolated healthy (cadaveric) liver. It is also shown that the cells distribute and / or migrate throughout the parenchyma and begin to organize and are maintained within the matrix as long as the experiment continues. The SEM images demonstrate that even at the microstructural level, the cellular organization of the cadaveric and recellularized matrices is similar.

[0138] Section E. Perfusion-Type vs. Immersion-Type Decellularization Example 1 - Decellularization Using Immersion Organs (rat liver, kidney, heart, lung, muscle, skin, bone, brain, and vasculature; porcine liver, gallbladder, kidney, and heart) were decellularized using the perfusion methods described herein.

[0139] The immersion methods described in U.S. Patent Nos. 6,753,181 and 6,376,244 were used to decellularize organs (rat liver, heart and kidney). Briefly, the organs were placed in dH 2 O and stirred at 4°C for 48 hours with a magnetic stir bar rotating at 100 rpm. After that, the organs were transferred to an ammonium hydroxide (0.05%) and Triton X-100 (0.5%) solution and the solution was stirred for an additional 48 hours with a magnetic stir bar (100 rpm). The solution was exchanged and the 48-hour immersion in ammonium hydroxide and Triton X-100 was repeated as many times as necessary to decellularize the organs (usually a visually acellular organ). This liver required approximately 5 repetitions of ammonium hydroxide and Triton X-100 to produce a visually acellular organ. After the decellularization process, the organs were placed in dH 2Transfer to O, stir for 48 hours (also stir at 100 rpm), and finally perform a final wash while stirring with PBS at 40°C.

[0140] Example 2 - Comparison of Perfusion-Type vs. Immersion-Type Figure 21A shows a photograph of a perfused decellularized porcine liver, and Figures 21B and 21C show SEMs of the vessels and parenchymal matrix of the perfused decellularized porcine liver, respectively. These photographs demonstrate the integrity of the vasculature and matrix of the perfused decellularized organ. On the other hand, Figure 22 shows an overall view of an immersion decellularized rat liver, in which matrix disruptions are visible at both low magnification (left) and high magnification (right).

[0141] Figure 23 shows SEMs of immersion decellularized rat livers (A and B), as well as perfused decellularized rat livers (C and D). These results clearly show that immersion decellularization significantly damaged the organ capsule (Glisson's capsule), while perfused decellularization retained the capsule. Furthermore, Figure 24 shows the tissue structures of immersion decellularized livers (A, HE staining; B, trichrome staining), and perfused decellularized livers (C, HE staining; D, trichrome staining). The immersion decellularized rat liver did not retain cells or dye during injection.

[0142] Figure 25 shows a comparison between immersion decellularization (upper row) and perfusion decellularization (lower row) of a rat heart. The photographs in the left column show the whole organs. As can be seen from the two photographs, the perfusion decellularized organ (lower left) is much more translucent than the immersion decellularized organ (upper left), and this immersion decellularized organ retains the iron-rich "reddish-brown" color of cadaveric muscle tissue and still appears to contain cells. The photographs in the middle column show the HE staining patterns of the decellularized tissues. From the staining, it is shown that after immersion decellularization (upper middle), numerous cells remain in both the parenchyma and the walls of the vascular system, while after perfusion decellularization (lower middle) (the vasculature of the present invention), it is clear that virtually all cells and cell debris have been removed. Furthermore, the scanning electron micrographs in the right column show that there are significant differences in the ultrastructure of the matrix between immersion (upper right) and perfusion (lower right) decellularization. Again, complete retention of cell components throughout fragments of myocardium was observed in all walls of the immersion decellularized heart, while in the perfusion decellularized heart these cell components were almost completely absent, as observed along with the retention of the spatial and structural features of the intact myocardium including the vasculature. For example, the perfusion decellularized matrix retained structural features including weave (w), coil (c), and muscle intersection (s) within the matrix, despite having completely lost cells.

[0143] Figure 26 shows a similar comparison performed using rat kidneys (immersion-based decellularization (upper), vs. perfusion-based decellularization (lower)). Unlike the heart, the immersion-based decellularized whole kidney (upper left) is very similar to the perfusion-based decellularized whole kidney (lower left) in that both are rather translucent. However, in the perfusion-based decellularized kidney, the network of vasculature within the perfusion-based decellularized organ is clearer and the branches are more visibly elevated compared to the immersion-based decellularized construct. Furthermore, the perfusion-based decellularized kidney retains the intact organ capsule, is surrounded by mesentery, and can be decellularized along with the accompanying adrenal gland as shown. The photographs in the middle column show the HE staining patterns of the two tissues. From the staining, it is shown that after immersion-based decellularization (upper middle), cell components and / or debris, and perhaps even intact nuclei (purple staining) remain, while after perfusion-based decellularization (lower middle), virtually all cells and / or all cell debris are shown to be removed. Similarly, the SEM photographs also demonstrate that the immersion-based decellularized kidney matrix (upper right) has received more damage than the perfusion-based decellularized kidney matrix (lower right). In the immersion-based decellularized kidney, surface "holes" or depressions are evident because the organ capsule is absent or damaged, while in the perfusion-based decellularized organ the capsule is intact.

[0144] Figure 27 shows SEM photographs of decellularized kidneys. Figure 27A shows a perfusion-based decellularized kidney and Figure 27B shows an immersion-based decellularized kidney. Figure 28A shows an SEM photograph of a perfusion-based decellularized heart and Figure 28B shows an SEM photograph of an immersion-based decellularized heart. Figure 29 shows an SEM photograph of an immersion-based decellularized liver. Furthermore, these images demonstrate the damage that immersion-based decellularization brings to the ultrastructure of the organ and the usability of the matrix after perfusion-based decellularization.

[0145] Other Aspects The present invention has been described in conjunction with the detailed description, but it will be understood that the above description is intended to be illustrative and not to limit the scope of the present invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. providing a perfused decellularized porcine liver or a perfused decellularized human liver, the decellularized liver comprising a decellularized extracellular matrix of the liver, the extracellular matrix comprising an outer surface, the extracellular matrix comprising a vascular tree retaining the morphology of the extracellular matrix prior to decellularization, a majority of fluids introduced into the vascular tree being retained, and the outer surface being intact; contacting the perfused decellularized liver with 40,000 or more regenerative cells under conditions in which the cells engraft, proliferate and / or differentiate in and on the decellularized liver to provide a recellularized liver having detectable liver-specific activity; A method for recellularizing a decellularized liver, comprising:

2. The method of claim 1, wherein the decellularized liver is contacted with 23 million or more regenerative cells.

3. The method of claim 1, wherein the decellularized liver is contacted with 30 million or more regenerative cells.

4. The method of claim 1, wherein the decellularized liver is contacted with 35 million or more regenerative cells.

5. The method of any one of claims 1 to 4, wherein the regenerative cells are hepatocytes.

6. The method of any one of claims 1 to 5, wherein the regenerative cells are injected into the decellularized liver via the portal vein.

7. The method of any one of claims 1 to 5, wherein the regenerative cells are injected into the decellularized liver.

8. 2. The method of claim 1, wherein the liver-specific activity comprises ureagenesis, albuminogenesis, or cytochrome p450 activity.

9. providing a decellularized porcine liver or lobe-containing portion thereof or a decellularized human liver or lobe-containing portion thereof, the decellularized liver or lobe-containing portion thereof comprising a decellularized extracellular matrix of the liver or lobe-containing portion thereof, the extracellular matrix comprising an outer surface, the extracellular matrix comprising a vascular tree retaining the morphology of the extracellular matrix prior to decellularization, a majority of fluids introduced into the vascular tree being retained, and the outer surface being intact; contacting a lobe of the decellularized liver or a lobe-containing portion thereof with a population of 40,000 or more regenerative cells under conditions in which the regenerative cells engraft, proliferate and / or differentiate within and on the decellularized liver lobe to provide a recellularized liver lobe or a lobe-containing portion thereof having detectable liver-specific activity; 1. A method for recellularizing a decellularized liver lobe, comprising:

10. The method of claim 9, wherein the regenerative cells are primary hepatocytes.

11. 11. The method of claim 9 or 10, wherein the regenerative cells are injected into the lobe via the portal vein.

12. 10. The method of claim 1 or 9, wherein the liver is a pig liver.

13. The method of claim 1 or 9, wherein the liver is a human liver.

14. 10. The method of claim 9, wherein the liver-specific activity comprises ureagenesis, albuminogenesis, or cytochrome p450 activity.

15. 10. The method of claim 1 or 9, wherein the regenerative cells are progenitor cells.

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